The present invention relates to the area of cancer diagnostics and therapeutics. More specifically, the invention relates to the isolation and purification of an early cancer detection marker protein of epithelial cells and the cloning of the DNA sequence encoding the protein. The invention further relates to the protein and DNA sequence for detecting and diagnosing individuals predisposed to cancer. The present invention also relates to therapeutic intervention to regulate the expression of the gene product.
Lung cancer is the most frequent cause of cancer death of both males and females in the United States, accounting for one in three cancer deaths(1). In the last thirty years, cancer-related survival of this disease has improved only minimally. Successful treatment of this disease by surgical resection and drug chemotherapy is strongly dependent on identification of early-stage tumors. A conceptually attractive early detection approach is to establish the presence of a cancer by evaluation of shed bronchial epithelial cells. In the late 1960""s Saccomanno et al. proposed the use of sputum cytology to evaluate cytomorphologic changes in the exfoliated bronchial epithelium as a technique to enhance the early detection of lung cancer(2). However, clinical trials using combination chest X-ray and sputum cytology have not shown any decrease in cancer-related mortality(3).
In 1988, Tockman et al. reported a sensitive method for early lung cancer detection by immunostaining cells contained within sputum samples with two lung cancer-associated monocional antibodies(4). The basis for this approach was to identify early pre-neoplastic changes in cells shed from bronchial epithelium. The antibodies used in that study were mouse monoclonal IgG""s designated 703D4, disclosed in U.S. Pat. No. 4,569,788, and 624H12. In an analysis of the contribution of the individual monoclonal antibodies to early detection of lung cancer, 703D4 alone identified 20 of the 21 detected true positive cases (4; U.S. Ser. No. 08/152,881 which issued to U.S. Pat. No. 5,455,159 on Oct. 3, 1995). 624H12 has been shown to detect an oncofetal antigen which is the Lewis""-related portion of a cell-surface glycoprotein (Mulshine/Magnani). The antigen for 703D4 was unknown.
703D4 was developed by immunization using a whole tumor cell extract, coupled to keyhole limpet hemocyanin, and selection was based on discrimination amongst subtypes of lung cancer histological subtypes. Preliminary studies showed the 703D4 antibody recognized a protein expressed by most non-small cell lung cancer cells(5). Immunoprecipitation defined a protein of Mr greater than 31 kDa. Since 703D4 demonstrated the ability to selectively detect changes related to the development of cancer in shed bronchial epithelium from the proximal airways, the antigen recognized by 703D4 was purified in the present invention to determine its identity and explore its relationship to early lung cancer detection. The present invention uses a biochemical approach for identification of the epithelial protein from non-small cell lung tumor cells.
With cigarette smoking the entire human respiratory tract is exposed to potential carcinogens and is at increased risk for cancer development. This phenomenon has been called xe2x80x9cfield cancerizationxe2x80x9d (8). A variety of epithelial changes have been observed throughout the respiratory tract of both smokers and lung cancer patients (8,9), which may be part of the xe2x80x9cfieldxe2x80x9d effect. Saccomanno et al. (6) have demonstrated that centrally located squamous carcinomas of the lung develop through a series of identifiable stages, namely squamous metaplasia, squamous metaplasia with atypia (mild, moderate, marked), carcinoma in situ, and invasive carcinoma (6). These findings were confirmed by later animal and human studies (7). This cytomorphologic classification is useful in defining preneoplastic changes in the proximal region of the lung cancer xe2x80x9cfieldxe2x80x9d. However, comparable events preceding the other major lung cancer histologies, especially those arising in the peripheral lung (terminal and respiratory bronchioles, alveolar epithelium) are not well defined.
The expression of an epithelial protein in both neoplastic and nonneoplastic regions of distal human lung was investigated.
The present invention describes the isolation and identification of an epithelial protein which is an early marker for cancer. It is an object of the present invention to provide an isolated and purified epithelial protein, peptide, or variants thereof which are an early marker for lung cancer.
It is an object of the present invention to provide an isolated, purified DNA molecule or portion thereof comprising the coding sequence for an epithelial protein, peptide or variant thereof which is an early marker for cancer.
It is another object of the invention to utilize the isolated DNA, or RNA molecule or portion thereof encoding the epithelial protein which is an early marker for cancer to detect and diagnose the gene and alterations thereof in tissues and cells.
It is another object of the invention to provide nucleic acid probes for the detection of the gene or protein thereof encoding an epithelial protein which is an early marker for cancer.
It is still another object of the invention to provide a method for diagnosing human preneoplastic and neoplastic cells and tissues. In accordance with the invention, the method comprises isolating cells, tissues or extracts thereof from a human and detecting the gene or portion thereof encoding an epithelial protein which is an early marker for cancer or their expression products from the cells, tissue or extracts thereof, wherein detection of a quantitative increase in the gene or expression products indicates preneoplasia and neoplasia.
Another object of the invention is a method for detecting mutations of a gene encoding the epithelial protein which is an early marker for cancer, contained within clones expressing the gene recovered from cancer cell.
Another method for diagnosing human preneoplastic and neoplastic cells and tissues is by detecting post-translational modifications of the epithelial protein in the preneoplastic and neoplastic cells and tissue by immunoassays such as Western blot or immunoelectrophoresis using an antibody that is reactive with the epithelial protein, by two-dimensional electrophoresis or by reverse-phase HPLC.
It is yet another object of the invention to provide a method for monitoring the efficacy of a therapeutic intervention to arrest cancer progression.
It is a further object of the invention to provide a kit comprising oligonucleotides comprising a nucleic acid sequence from DNA, RNA or portion thereof encoding the epithelial protein which is an early marker for cancer, for use in the methods of diagnosis of cancer and early cancer and for use in methods of monitoring the efficacy of cancer treatment.
Still another object of the invention is to provide the epithelial protein, peptides or variants thereof which one substantially homologous to a portion of at least one heterogenous nuclear ribonucleotide protein for use in diagnostic and detection assays, in particular for immunoassays.
One object of the invention is an inhibitory protein analog of the epithelial protein which is capable of binding to the same binding site recognized by the epithelial protein on RNA. Such an analog is capable of competitively inhibiting the function of the epithelial protein, peptide or variant thereof in vitro and in vivo.
It is yet another object of the invention to provide a method for detecting susceptibility to cancer and for diagnosing early-onset tumorigenesis in mammalian cells and tissue. In accordance with the invention, the method comprises isolating a mammalian biological sample and detecting a nucleic acid sequence encoding an epithelial protein or portion thereof which is an early marker for cancer.
Yet another object of the invention is to provide a method of altering or downregulating the expression of the gene or portion thereof encoding an epithelial protein or portion thereof which is an early marker for cancer of epithelial cells which comprises introduction of antisense oligonucleotides which are substantially complementary to the gene in the epithelial cell. The antisense oligonucleotide allows for non-neoplastic growth of the epithelial cell.
Another object of the invention is to provide a method for screening for chemotherapeutic drugs and for monitoring the efficacy of a chemotherapeutic and intervention drugs.
It is a further object of the invention to provide a transgenic animal which has incorporated into its genome one or more copies of a nucleic acid sequence which encodes an epithelial protein which is an early marker for cancer. The incorporation of the nucleic acid sequence results in overexpression or expression of multiple forms or variants of the epithelial protein. The resulting transgenic animal is more prone to develop cancer and may develop cancer at an accelerated rate at one or more locations in the body. Such transgenic animals are useful for screening therapeutic drugs useful for treating or inhibiting cancer.
It is yet another object of the invention to provide an antibody reactive to an epithelial protein, peptide or variant thereof. Such antibodies are useful in diagnosis and treatment of cancer.